3T3L-1 induction

Dear researcher:
Our lab use the 3T3L-1 cell line for induction experiment. We use the DMEM+10% FBS medium containing 1ėg/ml insulin, 1ėM dexamethasone and 0.5mM 3-iso-butyl-1-methylxanthine for 48hr. Then change DMEM+10% FBS medium containing 1ėg/ml insulin for 48 hr. After that we change DMEM+10% FBS medium for 48 hr and harvest cells.
But recently the 3T3-L1 preadipocyte differentiation is much less than usual. Do anybody know what happen to the cell line? Or our induction protocol need to modify?
Please give me some suggession. Thanks a lot!

1) have you changed your serum lot recently ? This can have dramatic effects on cell response. Preadipocytes will undergo differentiation in serum alone and whatever factors are present will vary from lot to lot.

2) How many passages have your cells been through ? I have found certain cell lines that will loose their ability to differentiate over time.

3) When do you passage cells ? before confluence or at confluence. Again this will effect cells. If you keep waiting till confluence you may well be causing subpopulations of cells to differenitate out leaving you will cells that are less able to differentiate when you want them to.

4) I beileve that normally the 3T3-L1 cells are grown to post confluence before switching to differentiation medium. Are you doing this ?

Any or all (or none ) of these may reduce your cells ability to differentiate. I am aware of one lab who published several studies on the effect of a growth factor on a certain cell line. They stopped culturing the cells for a period and when they re-started have never been able to repeat their results !!!!